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Published in J. Environ. Qual. 33:1202-1209 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Bioremediation and Biodegradation

Coupled Abiotic–Biotic Mineralization of 2,4,6-Trinitrotoluene (TNT) in Soil Slurry

Paul S. Schradera and Thomas F. Hessb,*

a Department of Environmental and Chemical Engineering, Yale University, New Haven, CT 06520
b Center for Hazardous Waste Remediation and Research, University of Idaho, Moscow, ID 83844-0904

* Corresponding author (tfhess{at}uidaho.edu).

Received for publication July 31, 2003.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The explosive 2,4,6-trinitrotoluene (TNT) is a contaminant of soils and ground waters worldwide. To help alleviate such environmental contamination, we investigated a coupled abiotic–biotic treatment scheme for remediating TNT-contaminated soil in slurry solutions. Two types of soil were used (sandy and silt loam) to simulate different soils that might be found at actual sites. These soils were subsequently contaminated with 5000 mg kg–1 TNT. Mineralization of TNT was initially optimized for minimum reactant use (Fe3+ and H2O2) and maximum soil slurry percentage (percent solids) using modified Fenton reactions conducted in the absence of light followed by the addition of an uncharacterized aerobic biomass. Greater than 97% TNT degradation was observed under optimum reaction conditions for both soils. Using two optimum reactant concentrations for each soil, coupled abiotic–biotic reactions showed an increase in TNT mineralization, from 41 to 73% and 34 to 64% in the sandy soil (10 and 20% slurry, respectively, 1470 mM H2O2), and increases from 12 to 23% and 13 to 28% in the silt loam soil (5% slurry, 294 and 1470 mM H2O2, respectively). These results show promise in the use of combined abiotic–biotic treatment processes for soils contaminated with high concentrations of TNT.

Abbreviations: HPLC, high performance liquid chromatography • TNT, 2,4,6-trinitrotoluene • WAS, waste-activated sludge


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THE EXPLOSIVE TNT has been widely used in military ordnance since World War I (Hess et al., 1998). Although currently not manufactured in the USA, the past production, testing, and decommissioning of such ordnance has contributed to widespread environmental contamination at many current and former U.S. Department of Defense (DOD) sites as well as other sites located throughout the United States and Europe (Urbanski, 1964; Spalding and Fulton, 1988). More than 1200 explosives-contaminated sites have been identified by the DOD alone with almost 90% of these sites containing contaminated ground water (Schmelling and Gray, 1995). Soil concentrations of TNT vary widely from trace levels to 14000 mg kg–1, nearing explosive levels. Because TNT is acutely toxic to a wide array of animals including humans even in low concentrations (Watts, 1998), mutagenic in the Ames test (Won et al., 1976), and listed as a priority pollutant by the USEPA (Schuster and Gratzfeld-Huesgen, 1993), remediation of TNT-contaminated soils and ground waters is legally mandated.

To date, numerous methods for the remediation of waters contaminated with TNT and its degradative products have been studied with the most common approach being that of either chemically or biologically mediated treatment. Chemical treatment has focused on advanced oxidative processes (AOPs), while biological transformation has utilized both bacterial and fungal systems. The AOPs previously applied to the treatment of TNT include ozone-catalyzed decomposition of TNT (Lang et al., 1998), TiO2–mediated photocatalysis (Schmelling et al., 1996; Schmelling and Gray, 1993, 1995), and Fenton chemistry (Li et al., 1997a, 1997b).

The Fenton reaction was discovered by and named for H.J.H. Fenton in the late 1800s (Fenton, 1894) and further defined by Haber and Weiss (1934), who showed that the hydroxyl radical (·OH) was the primary reactive species. The classic Fenton reaction is initiated by adding dilute hydrogen peroxide to a degassed solution of Fe(II), resulting in nearly stoichiometric generation of hydroxyl radicals. Many environmental applications of Fenton chemistry involve modifications to the above-mentioned reaction, including use of higher concentrations of hydrogen peroxide, heterogeneous catalysts, or Fe(III). These conditions, although not as stoichiometrically efficient, are often necessary to treat sorbed contaminants in soils and ground water (Tyre et al., 1991). Because of their reactivity with many organic contaminants at or near diffusion-controlled rates (>109 M–1 s–1), hydroxyl radicals can destroy aqueous biorefractory compounds such as perchloroethylene, hexachlorocyclopentadiene, and hexachlorobenzene within minutes (Leung et al., 1992; Sato et al., 1993; Watts et al., 1994). Fenton's reagent has been used to treat TNT-contaminated aqueous solutions resulting in complete degradation within 8 h and greater than 40% mineralization within 24 h (Li et al., 1997a; Hess and Schrader, 2002).

Both anaerobic and aerobic biodegradation processes have been investigated for the destruction of TNT. Aerobic TNT biodegradation has recently been demonstrated; however, problems limit it as an effective process such as accumulation of metabolic intermediates (Vorbeck et al., 1994; Ramos et al., 1995), inhibitory intermediate compound formation (Michels and Gottschalk, 1995), and low mineralization (Fernando et al., 1990). Recent research has suggested that anaerobic biological processes probably hold the most promise for stand-alone bioremediation of TNT (Funk et al., 1993; Preuss and Rieger, 1995; Crawford, 1995; Regan and Crawford, 1994; Lewis et al., 1997). It has been pointed out, however, that the mineralization of TNT from anaerobic processes is not substantial although the parent compound can be entirely transformed to intermediary metabolites (Crawford, 1995).

The combination of chemical and biological treatment processes has been shown to have advantages over either process alone (Carberry and Benzing, 1991; Koyama et al., 1994; Scott and Ollis, 1995; Ravikumar and Gurol, 1991). In these studies, the authors investigated sequential processes using abiotic reactions as a pretreatment step for a separate, follow-on biological reaction. Our research into sequential, coupled processes has indicated that either TiO2–mediated photocatalysis or Fenton's reagent followed by biological degradation (Hess et al., 1998; Hess and Schrader, 2002) as well as coexistent abiotic and biotic transformations (Buyuksonmez et al., 1998, 1999; Howsawkeng et al., 2001) can be used to treat biorecalcitrant compounds.

Based on the results of our previous work (Hess and Schrader, 2002), we determined that a combined chemical and biological treatment process should be effective for remediating TNT-contaminated soils. Our present objectives were to (i) determine the efficacy of modified Fenton chemistry to remediate TNT-contaminated soil, (ii) use an activated sludge biomass to promote the degradation of products resulting from the abiotic treatment, and (iii) optimize the kinetics of TNT degradation and mineralization using the coupled abiotic and biotic reactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Chemical Reagents
Iron (III) sulfate pentahydrate (97%) was purchased from Aldrich Chemical Company (Milwaukee, WI); reagent-grade H2O2 (30% v/v) was obtained from J.T. Baker (Phillipsburg, NJ); and TNT (99%) was purchased from Chem Service (West Chester, PA). Ecolite (+) scintillation cocktail was purchased from ICN Biomedicals (Costa Mesa, CA). Uniformly ring labeled TNT with a specific activity of 2.18 MBq mM–1 (>99%) was synthesized by Dr. Stefan Goszczynski of the Environmental Biotechnology Institute, University of Idaho (Moscow, ID). All other chemicals were of the highest available purity and double-deionized water (>18 M{Omega}-cm) was used to prepare solutions.

Experimental Design
Three types of experiments were conducted and described in this paper. Initial experimentation, for abiotic process optimization, was conducted to find maximal TNT mineralization extent using minimal reactant concentrations. The TNT degradation extent was also measured during this experimentation. Subsequent kinetic experiments, measuring TNT mineralization over time for the coupled abiotic–biotic process, were then conducted based on optimal reactant conditions found previously. A separate set of experiments was conducted to measure the effect of the coupled treatment process on bacterial numbers in the soils tested.

Optimization experiments were based on a factorial experimental design. The three-level design (Table 1) included H2O2 and soil slurry concentrations as experimental variables and subsequent biomass addition with either TNT degradation or mineralization as the response. Hydrogen peroxide and soil slurry percentages were tested over ranges of 59 to 1470 mM and 5 to 30%, respectively. We defined optimization within a narrow concentration range of H2O2 to avoid undue process problems, foaming or heating, and soil sterilization that can occur with extremely high concentrations of peroxide addition. Optimal reaction conditions, lowest concentration of hydrogen peroxide, and highest slurry percentage were determined by the greatest extent of TNT mineralization in the coupled abiotic–biotic system (Table 1). The optimal reaction conditions were then used for the subsequent kinetic studies.


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  		Table 1. TNT degradation and mineralization in modified Fenton reactions as affected by soil slurry and hydrogen peroxide concentrations and secondary treatment with activated sludge biomass. Experiments were conducted without light on both sandy and loam silt soils (Southwick series) containing 5000 mg kg–1 TNT.{dagger}

 
TNT Destruction in Soil Slurry
Experiments were conducted with two regional soils from the Southwick series (fine-silty, mixed, superactive, mesic Oxyaquic Argixerolls) (Table 2), a silt loam and a sand, collected from a University of Idaho site at approximately 30 and 100 cm below the surface, respectively. The soils were not sieved nor homogenized although clumps greater than 1 cm were not used and all plant debris was removed. The soils were dried at 60°C overnight before use.


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  		Table 2. Characteristics of soils used in all experiments.

 
Soils (5 g) were contaminated with solutions of TNT (5000 mg kg–1) in methanol using either nonlabeled TNT for degradation analysis or mixtures of nonlabeled TNT and enough 14C-UL-TNT to provide approximately 1.67 x 103 Bq per flask for mineralization analysis. Once the methanol evaporated, water was added to each flask to obtain the desired soil slurry percentage. No pH adjustments were made to the soil slurries. Soluble Fe(III) sulfate-pentahydrate (5 mM as Fe3+) was added to the reaction solution in a concentrated aqueous form such that the subsequent addition of 30% hydrogen peroxide combined with the previously added water volumes resulted in the various concentrations shown in the experimental design tables (Table 1). The concentration of iron was found to be optimal in preliminary soil studies (data not shown) and in our previous study of aqueous systems (Hess and Schrader, 2002). Fenton reactions were initiated by the addition of the appropriate amount of hydrogen peroxide. The abiotic Fenton reactions were allowed to proceed for 48 h after initiation. For samples receiving coupled abiotic–biotic treatment, activated sludge biomass obtained from the Moscow, ID, wastewater treatment plant was added at a concentration of 467 mg L–1 after neutralization of the soil solution pH, similar to previously described work (Hess and Schrader, 2002).

TNT Degradation Analyses
For analyses of TNT degradation, soil slurry subsamples from each optimization experiment were taken and filtered through paper filters, and the remaining soil solids extracted with 25 mL of acetonitrile. One-milliliter samples of either the aqueous filtrate or soil extract were prepared for high performance liquid chromatography (HPLC) analysis by filtering through a 0.2-µm nylon filter and placed into 1.5-mL amber vials with Teflon-lined septa. TNT concentrations were then determined using HPLC (Model 1090, Series II; Hewlett-Packard, Palo Alto, CA) equipped with a C18 ODS guard column connected to a 250- x 2.0-mm x 5-µm, C18, reverse-phase column (Phenomenex, Torrance, CA). A binary solvent, gradient elution methodology was used and consisted of (i) acetonitrile and (ii) 0.5 mM lithium phosphate buffer, pH 4.0 ± 0.1, at a flow rate of 0.22 mL min–1. Initial conditions were 5% acetonitrile (0–3 min), to 51% acetonitrile (3–21 min, held 12 min), to 70% acetonitrile (21–29 min, held 4 min), with a return to initial setup conditions by 32 min. A 10-µL injection of each sample collected was analyzed with the temperature of the HPLC column held constant at 40°C. The HPLC was equipped with a diode array UV/visible light detector (DAD) monitoring A230 with continuous scanning of the absorption spectrum of each peak from 190 to 600 nm. Compounds detected were identified by a comparison of their retention times and UV/visible light spectra with those of authentic standards. Results were corrected for TNT extractability.

TNT Mineralization Analysis
TNT mineralization was measured in both optimization experiments and kinetic experiments by capture and quantification of 14CO2 produced during the modified Fenton reactions. All reactions were conducted in 500-mL biometer flasks (Code of Federal Regulations, 1996) covered with aluminum foil. Each biometer flask was sealed with a rubber stopper and containing a glass cup (holding base solution) suspended in the atmosphere of the flask and a piece of glass tubing extending from the atmosphere of the flask through the stopper. Attached to the outside end of the glass tubing was an expandable bladder used to hold the gasses evolved from the reaction and allow free exchange with the flask atmosphere. The glass cup contained 1 mL of a 0.1 M NaOH solution used to capture CO2 from the flask atmosphere. For optimization experiments, the 1-mL NaOH sample was collected, as well as two successive 1-mL H2O rinsates, at the end of each treatment. For kinetic experiments, the sample and rinsates were collected at timed intervals during the course of the experiment. All samples were added directly to 15 mL of Ecolite (+) liquid scintillation cocktail and analyzed by scintillation counting as described below to quantify the amount of 14CO2 generated from the reaction. Knowing the original quantity of 14C-TNT in the slurry, we determined the extent (percent) of TNT mineralization. The soil slurries were filtered through a paper cone filter. One milliliter of the filtrate was then either added to 15 mL of scintillation cocktail for radioisotope analysis or filtered through a 0.2-µm nylon filter in preparation for HPLC analysis (as described above). The filters, with soil solids, were then extracted with 25 mL of acetonitrile on an orbital shaker at 220 rpm for 24 h. One milliliter of the extract was analyzed either by liquid scintillation counting or HPLC.

All samples from mineralization studies were counted with a liquid scintillation analyzer (Tri-Carb Model 2100TR; Packard Bioscience/PerkinElmer, Wellesley, MA) using a 14C protocol. The protocol subtracted counts of 14C from a blank matrix (NaOH) to avoid sample bias due to background enhancement radioactivity.

Most Probable Number Analyses
A most probable number (MPN) analysis was used to determine the concentration of bacteria present in soil samples both before and after soil treatments. All tests were performed in duplicate. Five grams of soil, either sandy or silt loam, was added to test tubes containing 50 mL of potassium phosphate buffer (the 1 x 100 dilution). The tubes were vortexed to separate bacteria from the soil and suspend them in the buffer. Serial dilutions from 1 x 101 to 1 x 106 were made in sterile phosphate buffer solution by mixing 1 mL of the first suspension with 9 mL of buffer. One-tenth of a milliliter from each of the last six dilutions (101–106) was plated onto sterile agar media as were the controls from dilutions using sterilized soil as the inoculum. Trypticase soy agar (TSA) alone, and plates containing Fenton reaction products of TNT degradation (0.1 normal) were used as the solid growth media for the bacteria. Plates were incubated at 30°C in a growth chamber. Growth was monitored for several days until colonies appeared (usually about 1.5–2 d) Plates with 30 to 300 colonies were counted with a plate counter to calculate the number of colony forming units (CFU) in the original soil sample. These numbers were used to determine the total number of bacteria present in the original samples.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
TNT Degradation
The maximum TNT degradation extent (nearly 98%) in the sandy soil corresponded to reaction conditions that produced the greatest extent of TNT mineralization (Table 1). In the silt loam soil, extent of TNT degradation increased with increasing hydrogen peroxide concentrations and decreased with increasing soil slurry percentage. A maximum degradation of nearly 98% in the silt loam soil also occurred in samples with the greatest total extent of TNT mineralization (Table 1). Degradation products observed in the aqueous extracts included 2,4,6-trinitrobenzoic acid and 1,3,5-trinitrobenzene (data not shown), similar to work of others (Li et al., 1997a).

TNT Mineralization Optimization
As can be seen in the results of process optimization testing (Table 1), the extent of TNT mineralization increased with increasing hydrogen peroxide concentrations in the abiotic experiments on the sandy soil. However, the maximum mineralization value, approximately 35%, did not occur at the lowest slurry percentage as was expected, but rather was found between the extreme values. This event may have been due to competitive reactions between the reactive oxygen radical species and excess Fe in mineral form on the soil at higher slurry concentrations. Others have shown Fenton-like reactions between peroxide and the native iron minerals in the soil for use in oxidation of soil contaminants (Tyre et al., 1991; Watts et al., 1994, 1997). Results of the analyses of the sandy soil used in our work (Table 2) indicated that iron minerals were present and presumably effective catalysts in the Fenton-like reactions. Maximum TNT mineralization in the coupled abiotic–biotic process in the sandy soil occurred at the same reaction conditions as for abiotic optimization (Table 1).

In all of the abiotic treatments of the silt loam soil, the extent of TNT mineralization decreased with both increasing slurry percentage and hydrogen peroxide concentration. Not surprisingly, the soil slurry percentage had the biggest effect on overall mineralization, probably due to the high organic carbon content of the soil solids and their quenching effect on reactive radicals or the previously mentioned competitive reactions for hydroxyl radical by iron in the soil matrix. Others have shown soil slurry percent to be a major process variable when treating contaminated soils with Fenton reagent (Spenser et al., 1996).

Maximum TNT mineralization in the coupled abiotic–biotic process in the silt loam soil occurred at different reaction conditions than for abiotic optimization (Table 1), notably at a higher peroxide concentration. Presumably, the higher peroxide concentration produced more biologically labile degradation products in the lowest soil slurry percentage with a concomitant increase in both biological and total mineralization.

Coupled Abiotic–Biotic TNT Mineralization Kinetics
We conducted kinetic studies on the abiotic mineralization of TNT in modified Fenton reactions of two sandy soil slurries, one 10% slurry with 1470 mM H2O2 and the other a 20% slurry also with 1470 mM H2O2. The cumulative TNT mineralization achieved in each reaction condition, 41 and 34%, respectively, was essentially complete within 24 h (Fig. 1). Both of these mineralization extents were greater than those found in the optimization studies but were within experimental error, especially considering the heterogeneity of such soil systems.



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  		Fig. 1. Mineralization of TNT (5000 mg kg–1) in modified Fenton reactions comparing abiotic with coupled abiotic–biotic treatments in sandy soil with (A) 10% soil slurry solution and 1470 mM H2O2 and (B) 20% soil slurry solution and 1470 mM H2O2. Abiotic reaction alone (•); abiotic + biotic reaction ({circ}). Arrow indicates time of application of biomass. Error bars on symbols indicate standard error of means (n = 3, p = 0.05); where absent, bars fall within symbols. All biotic reactions were conducted after solution neutralization.

 
In an effort to increase TNT mineralization using a coupled abiotic–biotic scheme, experiments were conducted with the addition of biotic treatment after the abiotic, modified Fenton treatments described above. A waste-activated sludge (WAS) biomass cultivated in a bench-scale sequencing batch reactor (Hess et al., 1993) was tested. Overall TNT mineralization increased from 41% (10% soil slurry, sandy soil) to 73% in the coupled abiotic–biotic system with WAS addition. Similarly, the extent of TNT mineralization increased from 34% (20% soil slurry, sandy soil) to 64% with the addition of biomass (Fig. 1).

A similar pattern of results to those above was observed in the kinetic studies performed on the silt loam soil, one 5% slurry and 294 mM H2O2, the other 5% slurry and 1470 mM H2O2. The total extent of TNT mineralization from the abiotic reaction under each condition was 12 and 13%, respectively (Fig. 2). These reactions were complete within 36 h, taking 12 h longer than those performed on the sandy soil. TNT mineralization in each case was somewhat greater than observed in the optimization experiments but again may be explained by the variability in soil heterogeneity. The follow-on treatment of these soils with WAS biomass again produced statistically significant increases in total mineralization to 23 and 28%, respectively (Fig. 2).



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  		Fig. 2. Mineralization of TNT (5000 mg kg–1) in modified Fenton reactions comparing abiotic with coupled abiotic–biotic treatments in silt loam soil with (A) 5% soil slurry solution and 294 mM H2O2 and (B) 5% soil slurry solution and 1470 mM H2O2. Abiotic reaction alone (•); abiotic + biotic reaction ({circ}). Arrow indicates time of application of biomass. Error bars on symbols indicate standard error of means (n = 3, p = 0.05); where absent, bars fall within symbols.

 
Analyses of 14C for all kinetic experiments indicated adequate carbon balances; 94.0 ± 2.5 and 94.4 ± 2.3% for sandy soils, abiotic and coupled abiotic–biotic experiments, respectively, and 73.1 ± 4.6 and 72.7 ± 3.9% for the silt loam soil, abiotic and coupled abiotic–biotic experiments, respectively. Lower recovery percentages (<80%) in some experiments were attributed to experimental error and the difficulties associated with soil analysis such as soil heterogeneity. It is also possible that the lower extent of mineralization in the silt loam soil, as compared with the sandy soil, may have been due in part to the lower recovery of total radioactivity, with sorption–sequestration and quenching in the soil matrix accounting for the remainder.

TNT mineralization rates and reaction efficiencies were calculated (Table 3) based on data in Fig. 1 and 2. The rate of TNT destruction was greatest in all abiotic reactions compared with biotic reactions. Abiotic TNT destruction rates were greater in the sandy soil as compared with the silt loam suggesting that competing reactions, such as with soil organic matter, played an important role in TNT destruction rate. We (Hess and Schrader, 2002) and others (Li et al., 1997a) have found carboxylic acids as degradation products from abiotic TNT destruction in aqueous solution and soil slurries, respectively. Calculations were also performed to indicate the efficiency of the reaction based on amount of TNT destroyed per unit of hydrogen peroxide used (Table 3). Examination of efficiency calculations for the silt loam samples indicated that significant increases in hydrogen peroxide concentration, while yielding minimal increases in TNT mineralization, actually resulted in a net decrease in reaction efficiency. While the reaction rates were slower in the biotic reactions as compared with the abiotic reactions, the overall reaction efficiencies were improved between 79 and 100% by the addition of biomass for each reaction condition.


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  		Table 3. Rate of TNT mineralization and reaction efficiency for modified Fenton reactions with two soils and abiotic, biotic, and coupled abiotic–biotic reactions.

 
The increase in the overall extent of TNT mineralization due to the addition of biomass was similar to results we obtained in aqueous systems (Hess and Schrader, 2002) but smaller in magnitude while requiring higher concentrations of hydrogen peroxide. This decrease in extent of mineralization and increased peroxide requirement was probably due to both the higher concentration of TNT being explored and the interactions of the soil matrix with the Fenton reaction, effectively quenching hydroxyl radicals. However, the mechanism of increased TNT mineralization in the coupled abiotic–biotic system over that of the abiotic reaction alone was probably due to biotic assimilation of the abiotic TNT transformation products. As reported by others (Li et al., 1997a), dicarboxylic acids, principally oxalic acid, and nitrate were the primary end products of Fenton reactions with TNT. An accumulation of oxalate and nitrate in aqueous solutions after abiotic reactions was previously reported (Hess and Schrader, 2002). Under such reaction conditions (low pH in the absence of light), oxalate would essentially have been nonreactive with hydroxyl radical (K·OH = 4.7 x 107 mol s–1; Buxton et al., 1988) in the presence of other substrates with higher reaction rates and would persist in solution. Several researchers have shown oxalate to be readily usable by many aerobic organisms, entering their metabolic pathways after breakdown via oxalate decarboxylase (Hokama et al., 2000; Tanner and Bornemann, 2000).

Soil Bacteria Analysis
An analysis was performed on both soils to determine the effect of contamination and abiotic and biotic treatments on the overall survival of soil bacteria. The sandy soil had a baseline population of 4.55 x 105 cells per gram of soil, which dropped to 3.55 x 104 cells g–1 as a result of contamination with TNT dissolved in methanol, determined using the most probable number (MPN) technique (Table 4). Similarly, the silt loam soil had an original population of 2.31 x 106 cells g–1 dropping to 6.55 x 105 cells g–1 post-contamination. Treatment of both soils with various modified Fenton reactions resulted in significant loss of the number of soil microorganisms detected. Others (Kastner et al., 2000) have found similar decreases in organism counts when practicing in situ Fenton treatment of soils. The lesser reduction of organisms in the sandy soil as compared with the silt loam soil after Fenton treatment may have been due to wasteful cycling of hydroxyl radical with abundant native iron (see Table 2) effectively quenching the radical attack on organic matter, including cells. As one would expect, the number of organisms found after biotic treatments was greater than originally present due to the high concentration of cells in the biomass used in the treatment process.


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  		Table 4. Bacterial analysis of the sandy and silt loam soils via the most probably number (MPN) technique before contamination with TNT, after contamination with TNT, and after the various treatments described in the text.

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Abiotic reactions were optimized for maximum TNT mineralization using a minimal amount of H2O2 at the greatest soil slurry concentration. Greater than 90% degradation was obtained under each reaction condition but mineralization varied widely. The two most optimal conditions in the sandy soil (10% slurry, 1470 mM H2O2 and 20% slurry, 1470 mM H2O2) yielded 41 and 34% TNT mineralization, respectively, through abiotic means only, while those in the silt loam soil (5% slurry, 294 mM H2O2, and 5% slurry, 1470 mM H2O2) were significantly lower, as expected, producing 12 and 13% mineralization, respectively.

Addition of biomass to the abiotic reaction products increased overall TNT mineralization extent in both the sandy and silt loam soils in the modified Fenton reactions. Using this coupled reaction process, abiotic TNT mineralization in the sandy soil was increased from 41 and 34% to 73 and 64%, respectively, using WAS biomass. The addition of WAS biomass to the silt loam soil slurries improved TNT mineralization from 12 and 13% to 23 and 28%, respectively. The increase in TNT mineralization was probably due to the abiotic formation of organic acids as intermediate products that were subsequently transformed by microorganisms.

Kinetics of the coupled abiotic–biotic reactions were relatively rapid as compared with conventional treatment methods. The abiotic reaction required 24 to 36 h while the addition of the biomass required an additional 3 d including the 12- to 24-h lag between the time of addition of the biomass and an increase in the mineralization of TNT. These kinetics are competitive with chemical treatments and much faster than most stand-alone biological treatments.

Reduction in native, cultivable, soil bacteria numbers due to Fenton reactions occurred in both the sand and silt loam soils. The greatest decrease in cell numbers, approximately six orders of magnitude, occurred in the silt loam soil at the highest peroxide concentration.

Our results indicate that a coupled abiotic–biotic treatment may be effective in treating TNT-contaminated soils. Further work on scale up of the processes to field-scale needs to be done before adoption of the treatment scheme for remediation of contaminated soils.


   ACKNOWLEDGMENTS
 
Appreciation is expressed to NSF EPSCoR Cooperative Agreement OSR-9350539 for funding assistance on this project.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 


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JEQ 2004 33: 1177-1182. [Full Text]  




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